DESCRIPTION: (Applicant's Abstract) Our previous work shows that embryonic amphibian spinal neurons exhibit spontaneous elevations of intracellular Ca2+ at early stages of development prior to synapse formation both in dissociated cell culture and in vivo. Further, our recent results indicate that these early Ca2+ transients specify key aspects of neuronal differentiation in a frequency encoded manner. These findings suggest that Ca2+ elevations initiate signal transduction cascades that determine subsequent steps of development. The proposed research has four specific aims. The 1st and 2nd Aims investigate the molecular regulation of neuronal phenotypes that are specified by spontaneous elevations of intracellular Ca2+. The 3rd and 4th Aims test hypotheses regarding regulation of differentiation by oscillations of second messengers, either at different developmental stages or via a different second messenger (cyclic AMP). The first aim investigates the hypothesis that particular frequencies of spontaneous Ca2+ spikes specify the cholinergic phenotype in spinal neurons, both in culture and in vivo. The second aim investigates the function of Ca2+ waves in axonal pathfinding in vivo, and analyzes the signal transduction cascade by which Ca2+ waves affect specific components of the growth cone cytoskeleton. The third aim is to investigate the role of Ca2+ transients in primary induction, which occurs well before terminal differentiation of neurons. The fourth aim is to investigate the interaction between elevations of cAMP and Ca2+ and the impact of this interaction on neuronal differentiation, and to analyze the incidence of spontaneous elevations of cAMP in embryonic neurons. A combination of embryological, imaging, electrophysiological, immunocytochemical and molecular biological techniques will be used to fulfill these aims. The immediate goal is to test hypotheses about specific mechanisms underlying differentiation of vertebrate spinal neurons in order to define the roles of transient elevations of second messengers in driving differentiation. The long term goal is to provide information about the cellular and molecular machinery that governs processes of development. It is expected that this work will contribute to an understanding of developmental disorders of the nervous system.